The spermatogonial stem cell (SSC), is an adult stem cell capable of both self-renewal and differentiation, thereby playing a critical role in spermatogenesis. Understanding the processes that govern SSC self-renewal and differentiation will provide essential mechanistic insight into the regulation of male fertility and is critical for future therapeutic treatment of male infertility in both animals and humans. In our ongoing efforts to understand the regulatory mechanisms governing SSC biology, we investigated the potential role of micro-RNAs (miRs), small RNA molecules that inhibit the translation of mRNA targets. It is well established that genes have an important role in regulating stem cells; however, little is known about miR regulation of the SSC. Abrogation of the miR processing protein, Dicer, in germ cells greatly reduces spermatogenesis, suggesting miRs are essential for the proliferation and differentiation of the SSC, but there have been no studies to demonstrate a direct function of specific miRs. Using high-throughput sequence analysis, we generated a miR profile unique to murine germ cells highly enriched for SSCs, which includes high levels of miR-21, and have shown that miR-21 expression contributes to SSC stemness. Moreover, we and others have demonstrated the importance of glial cell line-derived neurotrophic factor (GDNF) signaling in promoting SSC self-renewal and proliferation. Our hypothesis is that the contribution of miRs to SSC self-renewal is in part controlled by GDNF, and that the two processes of GDNF signaling and miR-target gene repression function in concert as part of a larger regulatory network mediating the fate of the SSC. Therefore, in Specific Aim 1, we will further evaluate the role of GDNF-induced transcription factor-dependent miR expression and its consequent effects on downstream target mRNAs in regulating GDNF signaling and SSC self-renewal, differentiation, and apoptosis. The multistage differentiative process of SSCs into spermatogonia and spermatozoa requires a multitude of extra- and intracellular signaling cues and we hypothesize that miRs also regulate the differentiation of germ cells. Therefore, in Specific Aim 2, we will use retinoic acid, a known inducer of germ cell differentiation, to evaluate the expression and function of miRs during early-stage spermatogenesis. Moreover, we will utilize innovative transgenic mouse lines specifically developed for these studies to track and quantitate the progress of SSCs towards differentiated spermatozoa. With these resources we can investigate the functional importance of miRs in the process of differentiation. We predict that these studies will transform the field in much the same way that our original studies on spermatogonial transplantation transformed our understanding of SSC biology. PUBLIC HEALTH RELEVANCE: The spermatogonial stem cell is an adult stem cell that undergoes both self-renewal and produces differentiated daughter cells that become spermatozoa, a process that is essential to spermatogenesis and is the foundation of male fertility. Infertility affects ~15% of couples, with nearly 50% being male-related and much of this involves defective spermatogenesis. The studies proposed are essential to understanding the regulation of spermatogenesis and causes of male infertility as well as for improvement of therapeutic treatment options.